ABSTRACT Once thought to be a mere consequence of the state of the cell, metabolism is now known to play a critical role in dictating cell differentiation. Non-oxidative glycolysis and oxidative phosphorylation (OxPhos) are the two sources of intracellular ATP. Factors that increase osteoblast activity and bone mass such as the Hypoxia- Inducible Factor 1a (HIF1) have been shown to activate non-oxidative glycolysis. In vitro osteogenic differentiation of mesenchymal progenitors increases both non-oxidative glycolysis and OxPhos. However, the role of OxPhos in osteoblast biology in vivo is largely unexplored. To fill this gap in knowledge, we generated a mutant mouse lacking Mitochondrial Transcription Factor A (TFAM) in uncommitted mesenchymal progenitors and their descendants (PRX;TFAMf/f). TFAM regulates transcription of the mitochondrial genes that encode thirteen subunits of the electron transport chain and thus controls OxPhos. Analysis of 3-week-old PRX;TFAMf/f bones revealed the presence of a severe low bone mass phenotype with spontaneous fractures in mutants. Our data thus indicate that mesenchymal TFAM is necessary for bone mass accrual. In addition, we provided preliminary evidence that loss of TFAM inhibits the in vitro differentiation of bone marrow stromal cells (BMSCs) into osteoblasts and significantly reduces their intracellular levels of ATP. Impairment of OxPhos is the most powerful, consistent and best characterized biological consequence of loss of TFAM across numerous cell types. However, TFAM also regulates duplication of mitochondrial DNA, and mitochondria have functions that go beyond OxPhos and ATP production. Therefore, to establish if the impairment of OxPhos and thus the decreased intracellular ATP is the primary cause of the PRX;TFAMf/f bone phenotype, we asked whether correcting the ATP levels through forced upregulation of non-oxidative glycolysis would prevent the low bone mass of PRX;TFAMf/f mice. For this purpose, we crossed PRX;TFAMf/f mutants with mice overexpressing a constitutively stabilized HIF1 in the same cells (PRX;HIF1dPAf/f). HIF1 is known to promote non-oxidative glycolysis and to impair OxPhos.. Preliminary analysis of PRX;TFAMf/f;HIF1dPAf/f double mutant mice revealed that increased HIF1 activity corrected the bone phenotype of PRX;TFAMf/f mutants. Building on these findings, we will now test the hypothesis that TFAM in cells of the osteoblast lineage is crucial for bone mass accrual and maintenance by promoting OxPhos and thus ensuring the proper levels of intracellular ATP. We will test our hypothesis in three Aims. Progressive impairment of mitochondrial activity has been associated with numerous aging-related diseases, but it is uncertain whether this association is due, at least in part, to a dysfunctional OxPhos. The successful accomplishment of the experiments we propose in this application will expand and deepen our knowledge of the role of energy metabolism, particularly OxPhos, in the regulation of osteoblast differentiation and bone mass accrual and maintenance.